In vitro invasion inhibition assay using antibodies against Plasmodium knowlesi Duffy binding protein alpha and apical membrane antigen protein 1 in human erythrocyte-adapted P. knowlesi A1-H.1 strain

Abstract

BackgroundThe rapid process of malaria erythrocyte invasion involves ligand–receptor interactions. Inducing antibodies against specific ligands or receptors that abrogate the invasion process is a key challenge for blood stage vaccine development. However, few candidates were reported and remain to be validated for the discovery of new vaccine candidates in Plasmodium knowlesi.MethodsIn order to investigate the efficacy of pre-clinical vaccine candidates in P. knowlesi-infected human cases, this study describes an in vitro invasion inhibition assay, using a P. knowlesi strain adapted to in vitro growth in human erythrocytes, PkA1-H.1. Recombinant proteins of P. knowlesi Duffy binding protein alpha (PkDBPα) and apical membrane antigen 1 (PkAMA1) were produced in Escherichia coli system and rabbit antibodies were generated from immune animals.ResultsPkDBPα and PkAMA1 recombinant proteins were expressed as insoluble and produced as a functional refolded form for this study. Antibodies against PkDBPα and PkAMA1 specifically recognized recombinant proteins and native parasite proteins in schizont-stage parasites on the merozoite organelles. Single and combination of anti-PkDBPα and anti-PkAMA1 antibodies elicited strong growth inhibitory effects on the parasite in concentration-dependent manner. Meanwhile, IgG prevalence of PkDBPα and PkAMA1 were observed in 13.0 and 46.7% in human clinical patients, respectively.ConclusionThese data provide support for the validation of in vitro growth inhibition assay using antibodies of DBPα and AMA1 in human-adapted P. knowlesi parasite PkA1-H.1 strain.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2420-4) contains supplementary material, which is available to authorized users.

Cross-species reactivity of antibodies against Plasmodium vivax blood-stage antigens to Plasmodium knowlesi

“…Recombinant protein expression was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma-Aldrich, St. Louis, MO). All recombinant proteins were solubilized, purified, and refolded as described elsewhere [47,54].…”

“…Meanwhile, P. knowlesi recombinant proteins were expressed for this study using wheat germ cell-free system for immunoscreening using specific primers (S2 Table), the procedures were described elsewhere [47]. Recombinant PkDBPα protein expression was described elsewhere [47].…”

“…Meanwhile, P. knowlesi recombinant proteins were expressed for this study using wheat germ cell-free system for immunoscreening using specific primers (S2 Table), the procedures were described elsewhere [47]. Recombinant PkDBPα protein expression was described elsewhere [47]. The DNA fragment encoding PkRhopH2 was amplified from P. knowlesi A1-H.1 genomic DNA with primer pair PkRhopH2_F (ggtcgcggatccgaattcATGGAGTTAGGCCA TACCGTG) and PkRhopH2_R (ggtggtggtgctcgagCTTCTCGATGTCTTCGTAGTCCA).…”

“…All P. vivax blood-stage antigen-specific antibodies were obtained during our previous studies (Table 1, S3 Table). Total IgG was purified from 1 mL of rabbit serum by using a protein G HP column according to the manufacturer's protocol (GE Healthcare Life Sciences) as described elsewhere [47]. All animal experimental protocols were approved by the Institutional Ethics Committee and followed the Ethical Guidelines for Animal Experiments of Kangwon National University (KIACUC-16-0158).…”

Untitled

“…Culture of P. knowlesi: Since the maintenance of P. knowlesi in-vitro in rhesus RBCs for several intraerythrocytic cycles was achieved in 1945, 27 attempts to develop a continuous in-vitro culture for this malaria parasite have never ended 28 . The long-term in-vitro culture system would allow full analysis of parasite-host cell interactions 29 , improve understanding of the biology of P. knowlesi such as the mechanism of invasion 30,31 and resistance markers 32 , and contribute to the development of new anti-malarial drugs and vaccines 31,33,34,35 . The study by Ball et al, (1945) indicated that target cells of P. knowlesi are not restricted to a certain age in macaques, however, in humans, it was found that P. knowlesi mainly invades reticulocytes 17,27 .…”